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目的观察犬骨髓来源成体祖细胞(MAPC)与软骨细胞共培养后其向软骨细胞定向分化的情况。方法采用全骨髓直接接种法体外培养犬MAPC,观察形态学特征、生长情况及活细胞数,绘制生长曲线,流式细胞仪检测SSEA-1、CD13、CD44、CD45、CD133、MHCⅡ表达率。采用软骨培育法原代培养兔软骨细胞并鉴定。实验组及对照组分别采用软骨细胞及培养基与MAPC在Transwell小室共培养,观察2组MAPC分化为软骨细胞的百分率,RT-PCR检测聚集蛋白多糖和Ⅱ型胶原mRNA含量,组间比较采用单因素方差分析。结果 MAPC原代培养呈多角形,8 d后呈克隆样生长,形态呈长条形或梭形;生长曲线显示MAPC接种1 d后开始增殖,6 d达到高峰,随后进入平台期;流式细胞仪检测结果显示细胞表面SSEA-1和CD13阳性,CD44、CD45、CD133、MHCⅡ阴性;实验组及对照组Ⅱ型胶原抗体阳性细胞比例分别为(26.1±4.5)%,对照组为(7.2±2.2)%,差异有统计学意义(P<0.05),RT-PCR显示实验组聚集蛋白多糖和Ⅱ型胶原mRNA含量分别为6.61±0.34、8.37±0.79,显著高于对照组1.46±0.51、2.14±0.48(P<0.05)。结论 MAPC与软骨细胞共培养可诱导其向软骨细胞分化。
Objective To observe the directional differentiation of canine bone marrow-derived adult progenitor cells (MAPCs) into chondrocytes co-cultured with chondrocytes. Methods The canine MAPC was cultured in vitro by direct inoculation of whole bone marrow. Morphological characteristics, growth and number of viable cells were observed. The growth curve was drawn. The expression of SSEA-1, CD13, CD44, CD45, CD133 and MHCⅡ were detected by flow cytometry. Rabbit chondrocytes were cultured and identified by cartilage culture. The experimental group and the control group were cultured with MAPCs in Transwell chambers respectively with chondrocytes and culture medium. The percentage of MAPCs differentiated into chondrocytes in two groups was observed. The content of aggrecan and type II collagen mRNA was detected by RT-PCR. Factor analysis of variance. Results Primary culture of MAPCs was polygonal. After 8 days, MAPCs grew in a clonal pattern with elongated or fusiform shape. The growth curve showed that MAPCs began to proliferate after 1 day of inoculation and peaked on the 6th day, then entered the plateau phase. Flow cytometry The results showed that the positive rates of type II collagen antibody positive cells in the experimental group and the control group were (26.1 ± 4.5)%, respectively, and those in the control group were (7.2 ± 2.2) )%, The difference was statistically significant (P <0.05). RT-PCR showed that the content of aggrecan and type Ⅱ collagen mRNA in experimental group were 6.61 ± 0.34,8.37 ± 0.79, which was significantly higher than that in control group (1.46 ± 0.51,2.14 ± 0.48 (P <0.05). Conclusions MAPC co-cultured with chondrocytes can induce its differentiation into chondrocytes.